A technique was developed by which polyacrylamide gel electrophoresis is utilized to detect basic ribonuclease activity rather than simply the presence of protein. Polyacrylamide gels containing low concentrations of a polynucleotide substrate were prepared. Spermine, present in high amounts during electrophoresis, enabled the basic RNases to migrate in the presence of polyanions such as a polyuridylic acid (poly(U)). Electrophoresis of the RNases under conditions of low pH and incubation of the gel at neutral pH followed by staining result in a colored gel containing clear bands which define regions of enzyme activity. This method (polynucleotide-polyacrylamide gel electrophoresis) is at least 10 to the 4th power times more sensitive for the detection of RNase activity than conventional electrophoresis followed by staining for protein. Ten picograms of bovine pancreatic RNase A, for example, could be visualized in a reproducible manner on polynucleotide containing gels. Using this technique, with a total of four different substrates in seventeen gels, it was possible to establish simultaneously that each of three enzymes purified from bovine muscle was distinct from the remaining two, that each was homogeneous, and that one of these RNases was identical to RNase A. Negligible quantities of enzyme were required for these analyses.